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为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。
In order to clarify the resistance effect of the broad-spectrum antiviral gene pac1 gene to Grapevine virus B (GVB), the pac1 gene was introduced into western tobacco 37B by Agrobacterium tumefaciens-mediated genetic transformation and the transgenic plants were subjected to PCR Identification and Southern blot analysis, virus infection by FV vaccination and real-time fluorescent quantitative RT-PCR detection of virus content in plants, and preliminary identification of transgenic plants. The results showed that the target gene Pac1 was successfully introduced and integrated into Western tobacco 37B genome, and a total of 10 transgenic lines were obtained. The percentage of positive plants in T1 lines of different lines was 16.7% -72.7%, indicating that the target gene could be successfully passed on to offspring. The transgenic plants generally delayed the onset of disease after vaccination, but the late symptoms were similar to the non-transgenic controls, of which only one transgenic line, B6, exhibited resistance to typical symptoms. In the detection of the virus content in the inoculated plants, all the transgenic plants detected the presence of the virus, but the virus content of the resistant B6 strain was significantly lower than that of the non-transgenic control, indicating that the transgenic plants did not completely resist the GVB infection. However, With disease resistance.